Evolution of aldolase antibodies in vitro: correlation of catalytic activity and reaction-based selection

Aldolase antibodies that operate via an enamine mechanism were developed by in vitro selection. Antibody Fab phage display libraries were created where the catalytic active site residues of aldolase antibodies 38C2 and 33F12 were combined with a naive human antibody V gene repertoire. Selection from these libraries with 1,3-diketones covalently trapped the amino groups of reactive lysine residues by formation of stable enaminones. The selected aldolase antibodies retained the essential catalytic lysine residue and its function in altered and humanized primary antibody structures. The substrate specificity of the aldolase antibodies was directly related to the structure of the diketone used for selection. The k(cat) values of the antibody-catalyzed retro-aldol reactions were correlated with the K(d) values, i.e. the reactivities of the selected aldolase antibodies for the corresponding diketones. Antibodies that bound to the diketone with a lower K(d) value displayed a higher k(cat) value in the retro-aldol reaction, and a linear relationship was observed in the plots of logk(cat) versus logK(d). These results indicate that selections with diketones directed the evolution of aldolase antibodies in vitro that operate via an enamine mechanism. This strategy provides a route to tailor-made aldol catalysts with different substrate specificities.     

Magnesium influences the discrimination and release of ADP by human RAD51

hRAD51 lacks cooperative DNA-dependent ATPase activity and appears to function with 5-10-fold less Mg2+ compared to RecA. We have further explored the effect of Mg2+ on adenosine nucleotide binding, ATPase, and DNA strand exchange activities. hRAD51 was saturated with the poorly hydrolyzable analog of ATP, ATPgammaS, at approximately 0.08 mM Mg2+. In contrast, > 0.5 mM Mg2+ was required to saturate hRAD51 with ADP. We found ADP to be a significantly less effective competitive inhibitor of the hRAD51 ATPase at low Mg2+ concentrations (0.08 mM). Mg2+ did not appear to affect the ability of ATPgammaS to competitively inhibit the hRAD51 ATPase. Low Mg2+ (0.08-0.12 mM) enhanced the steady-state ATPase of hRAD51 while higher Mg2+ concentration (> 0.3 mM) was inhibitory. At low Mg2+, hRAD51 appeared capable of nearly complete hydrolysis of available ATP, suggesting a lack of ADP product inhibition. There was a strong correlation between the amount of Mg2+ required for stable ADP binding and the inhibition of hRad51 strand exchange activity. Simultaneous inclusion of exogenous ATP and chelation of Mg2+ with EDTA significantly enhanced ADP-->ATP exchange by hRAD51. These studies are consistent with the hypothesis that Mg2+ influences the discrimination and release of ADP, which may sequentially impose an important regulatory step in the hRAD51 ATPase cycle.     

Systematically assessing the influence of 3-dimensional structural context on the molecular evolution of mammalian proteomes

The 3-dimensional (3D) structural context of amino acid residues in a protein could significantly impact the level of selective constraint on the residues. Here, by analyzing 767 mammalian proteins, we systematically investigate how various 3D structural contexts influence selective constraint. The structural contexts we examined include solvent accessibility, secondary structure, and intramolecular residue-residue interactions. Through this analysis, we offer quantitative information on how 3D structural contexts affect the level of selective constraint.     

Residue analysis of organophosphorus and organochlorine pesticides in fatty matrices by gas chromatography coupled with electron-capture detection

A multiresidue method for the simultaneous determination of 22 organochlorine (OCs) and organophosphorus (Ops) pesticides (including isomers and metabolites), representing a wide range of physicochemical properties, was developed in fatty matrices extracted from meat. Pesticides were extracted from samples with acetonitrile/n-hexane (v:v, 1:1). The analytical screening was performed by gas chromatography coupled with electron-capture detection (ECD). The identification of compounds was based on their retention time and on comparison of the primary and secondary ions. The optimized method was validated by determining accuracy (recovery percentages), precision (repeatability and reproducibility), and sensitivity (detection and quantitation limits) from analyses of samples fortified at 38 to 300 ng/g levels. Correlation coefficients for the 22 extracted pesticide standard curves (linear regression analysis, n = 3) ranged from 0.998 to 1.000. Recovery studies from 2 g samples fortified at 3 levels demonstrated that the GC-ECD method provides 64.4-96.0% recovery for all pesticides except 2,4'-DDE (44.6-50.4%), 4,4'-DDE (51.1-57.5%) and 2,4'-DDT (50.0-51.2%). Both repeatability and reproducibility relative standard deviation values were < 20% for all residues. Detection limits ranged from 0.31 to 1.27 ng/g and quantification limits were between 1.04 and 4.25 ng/g. The proposed analytical method may be used as a simple procedure in routine determinations of OCs and Ops in meat. It can also be applied to the determination of pesticide multi-residues in other animal products such as butter and milk.     

NSAID activated gene (NAG-1), a modulator of tumorigenesis

The NSAID activated gene (NAG-1), a member of the TGF-beta superfamily, is involved in tumor progression and development. The over-expression of NAG-1 in cancer cells results in growth arrest and increase in apoptosis, suggesting that NAG-1 has anti-tumorigenic activity. This conclusion is further supported by results of experiments with transgenic mice that ubiquitously express human NAG-1. These transgenic mice are resistant to the development of intestinal tumors following treatment with azoxymethane or by introduction of a mutant APC gene. In contrast, other data suggest a pro-tumorigenic role for NAG-1, for example, high expression of NAG-1 is frequently observed in tumors. NAG-1 may be like other members of the TGF-beta superfamily, acting as a tumor suppressor in the early stages, but acting pro-tumorigenic at the later stages of tumor progression. The expression of NAG-1 can be increased by treatment with drugs and chemicals documented to prevent tumor formation and development. Most notable is the increase in NAG-1 expression by the inhibitors of cyclooxygenases that prevent human colorectal cancer development. The regulation of NAG-1 is complex, but these agents act through either p53 or EGR-1 related pathways. In addition, an increase in NAG-1 is observed in inhibition of the AKT/GSK-3beta pathway, suggesting NAG-1 alters cell survival. Thus, NAG-1 expression is regulated by tumor suppressor pathways and appears to modulate tumor progression.     

An age-structured epidemic model of rotavirus with vaccination

The recent approval of a rotavirus vaccine in Mexico motivates this study on the potential impact of the use of such a vaccine on rotavirus prevention and control. An age-structured model that describes the rotavirus transmission dynamics of infections is introduced. Conditions that guarantee the local and global stability analysis of the disease-free steady state distribution as well as the existence of an endemic steady state distribution are established. The impact of maternal antibodies on the implementation of vaccine is evaluated. Model results are used to identify optimal age-dependent vaccination strategies. A convergent numerical scheme for the model is introduced but not implemented. This paper is dedicated to Prof. K. P. Hadeler, who continues to push the frontier of knowledge in mathematical biology.     

G-rich, a Drosophila selenoprotein, is a Golgi-resident type III membrane protein

G-rich is a Drosophila melanogaster selenoprotein, which is a homologue of human and mouse SelK. Subcellular localization analysis using GFP-tagged G-rich showed that G-rich was localized in the Golgi apparatus. The fusion protein was co-localized with the Golgi marker proteins but not with an endoplasmic reticulum (ER) marker protein in Drosophila SL2 cells. Bioinformatic analysis of G-rich suggests that this protein is either type II or type III transmembrane protein. To determine the type of transmembrane protein experimentally, GFP-G-rich in which GFP was tagged at the N-terminus of G-rich, or G-rich-GFP in which GFP was tagged at the C-terminus of G-rich, were expressed in SL2 cells. The tagged proteins were then digested with trypsin, and analyzed by Western blot analysis. The results showed that the C-terminus of the G-rich protein was exposed to the cytoplasm indicating it is a type III microsomal membrane protein. G-rich is the first selenoprotein identified in the Golgi apparatus.     

Determination of functional strength imbalance of the lower extremities

The purposes of this study were (a) to determine whether a significant strength imbalance existed between the left and right or dominant (D) and nondominant (ND) legs and (b) to investigate possible correlations among various unilateral and bilateral closed kinetic chain tests, including a field test, and traditional isokinetic dynamometry used to determine strength imbalance. Fourteen Division I collegiate women softball players (20.2 +/- 1.4 years) volunteered to undergo measures of average peak torque for isokinetic flexion and extension at 60 degrees .s(-1) and 240 degrees .s(-1); in addition, measures of peak and average force of each leg during parallel back squat, 2-legged vertical jump, and single-leg vertical jump and performance in a 5-hop test were examined. Significant differences of between 4.2% and 16.0% were evident for all measures except for average force during single-leg vertical jump between the D and ND limbs, thus revealing a significant strength imbalance. The 5-hop test revealed a significant difference between D and ND limbs and showed a moderate correlation with more sophisticated laboratory tests, suggesting a potential use as a field test for the identification of strength imbalance. The results of this study indicate that a significant strength imbalance can exist even in collegiate level athletes, and future research should be conducted to determine how detrimental these imbalances could be in terms of peak performance for athletes, as well as the implications for injury risk.